The present invention relates to a Type I interferon complex, composed of the polypeptide sequence of the human interferon xcex1/xcex2 receptor (IFNAR2) extracellular domain and a Type I interferon (IFNxcex1, IFNxcex2, and IFNxcfx89). Such a complex improves the stability, enhances the potency, and prolongs the pharmacokinetics in vivo of free IFN for anti-viral, anti-cancer and immune modulating activity. More particularly, the complex is a fusion protein, or a covalent complex, or a non-covalent complex containing the polypeptide sequence of the entire extracellular domain of IFNAR2, or any interferon-binding subfraction thereof, complexed to a Type I interferon (IFNxcex1, IFNxcex2, IFNxcfx89), or any biologically active subfraction thereof.
Interferons are classified either as the leukocyte and fibroblast derived Type I interferons, or as the mitogen induced or xe2x80x9cimmunexe2x80x9d Type II interferons (Pestka et al, 1987). Through analysis of sequence identities and common biological activities, Type I interferons include interferon alpha (IFNxcex1), interferon beta (IFNxcex2) and interferon omega (IFNxcfx89), while Type II interferon includes interferon gamma (IFNxcex3). The IFNxcex1, IFNxcex2 and IFNxcfx89 genes are clustered on the short arm of chromosome 9 (Lengyl, 1982). There are at least 25 non-allelic IFNxcex1 genes, 6 non-allelic IFNxcfx89 genes and a single IFNxcex2 gene. All are believed to have evolved from a single common ancestral gene. Within species, IFNxcex1 genes share at least 80% sequence identity with each other. The IFNxcex2 gene shares approximately 50% sequence identity with IFNxcex1; and the IFNxcfx89 gene shares 70% homology with IFNxcex1 (Weissman et al, 1986; Dron et al, 1992). IFNxcex1 has a molecular weight range of 17-23 kDa (165-166 amino acids), IFNxcex2, xcx9c23 kDa (166 amino acids) and IFNxcfx89, xcx9c24 kDa (172 amino acids).
Type I interferons are pleiotropic cytokines having activity in host defense against viral and parasitic infections, as anti-cancer cytokines and as immune modulators (Baron et al, 1994; Baron et al, 1991). Type I interferon physiological responses include anti-proliferative activity on normal and transformed cells; stimulation of cytotoxic activity in lymphocytes, natural killer cells and phagocytic cells; modulation of cellular differentiation; stimulation of expression of class I MHC antigens; inhibition of class II MHC; and modulation of a variety of cell surface receptors. Under normal physiological conditions, IFNxcex1 and IFNxcex2 (IFNxcex1/xcex2) are secreted constitutively by most human cells at low levels with expression being up-regulated by addition of a variety of inducers, including infectious agents (viruses, bacteria, mycoplasma and protozoa), dsRNA, and cytokines (M-CSF, IL-1xcex1, IL-2, TNFxcex1). The actions of Type I interferon in vivo can be monitored using the surrogate markers, neopterin, 2xe2x80x2, 5xe2x80x2 oligoadenylate synthetase, and xcex22 microglobulin (Alam et al, 1997; Fierlbeck et al, 1996; Salmon et al, 1996).
Type I interferons IFNxcex1/xcex2/xcfx89) act through a cell surface receptor complex to induce specific biologic effects, such as anti-viral, anti-tumor, and immune modulatory activity. The Type I IFN receptor (IFNAR) is a hetero-multimeric receptor complex composed of at least two different polypeptide chains (Colamonici et al, 1992; Colamonici et al, 1993; Platanias et al, 1993). The genes for these chains are found on chromosome 21, and their proteins are expressed on the surface of most cells (Tan et al, 1973). The receptor chains were originally designated alpha and beta because of their ability to be recognized by the monoclonal antibodies IFNxcex1R3 and IFNaRxcex21, respectively. Most recently, these have been renamed IFNAR1 for the alpha subunit and IFNAR2 for the beta subunit. In most cells, IFNAR1 (alpha chain, Uze subunit) (Uze et al, 1990) has a molecular weight of 100-130 kDa, while IFNAR2 (beta chain, BL, IFNxcex1/xcex2R) has a molecular weight of 100 kDa. In certain cell types (monocytic cell lines and normal bone marrow cells) an alternate receptor complex has been identified, where the IFNAR2 subunit (xcex2S) is expressed as a truncated receptor with a molecular weight of 51 kDa. The IFNAR1 and IFNAR2 xcex2S and xcex2L subunits have been cloned (Novick et al, 1994; Domanski et al, 1995). The IFNAR2 xcex2S and xcex2L subunits have identical extracellular and transmembrane domains; however, in the cytoplasmic domain they only share identity in the first 15 amino acids. The IFNAR2 subunit alone is able to bind IFNxcex1/xcex2, while the IFNAR1 subunit is unable to bind IFNxcex1/xcex2. When the human IFNAR1 receptor subunit alone was transfected into murine L-929 fibroblasts, no human IFNxcex1s except IFNxcex18/IFNxcex1B were able to bind to the cells (Uze et al, 1990). The human IFNAR2 subunit, transfected into L cells in the absence of the human IFNAR1 subunit, bind human IFNxcex12, binding with a Kd of approximately 0.45 nM. When human IFNAR2 subunits were transfected in the presence of the human IFNAR1 subunit, high affinity binding could be shown with a Kd of 0.026-0.114 nM (Novick et al, 1994; Domanski et al, 1995). It is estimated that from 500-20,000 high affinity and 2,000-100,000 low affinity IFN binding sites exist on most cells. Although the IFNAR1/2 complex (xcex1/xcex2s or xcex1/xcex2L) subunits bind IFNxcex1 with high affinity, only the xcex1/xcex2L pair appears to be a functional signaling receptor.
Transfection of the IFNAR1 and the IFNAR2 xcex2L subunits into mouse L-929 cells, followed by incubation with IFNxcex12, induces an anti-viral state, initiates intracellular protein phosphorylation, and causes the activation of intracellular kinases (Jak1 and Tyk2) and transcription factors (STAT 1, 2, and 3) (Novick et al, 1994; Domanski et al, 1995). In a corresponding experiment, transfection of the IFNAR2 xcex2S subunit was unable to initiate a similar response. Thus, the IFNAR2 xcex2L subunit is required for functional activity (anti-viral response) with maximal induction occurring in association with the IFNAR1 subunit.
In addition to membrane bound cell surface IFNAR forms, a soluble IFNAR has been identified in both human urine and serum (Novick et al, 1994; Novick et al, 1995; Novick et al, 1992; Lutfalla et al, 1995). The soluble IFNAR isolated from serum has an apparent molecular weight of 55 kDa on SDS-PAGE, while the soluble IFNAR from urine has an apparent molecular weight of 40-45 kDa (p40). Transcripts for the soluble p40 IFNAR2 are present at the mRNA level and encompass almost the entire extracellular domain of the IFNAR2 subunit with two new amino acids at the carboxy terminal end. There are five potential glycosylation sites on the soluble IFNAR2 receptor. The soluble p40 IFNAR2 has been shown to bind IFNxcex12 and IFNxcex2 and to inhibit in vitro the anti-viral activity of a mixture of IFNxcex1 species (xe2x80x9cleukocyte IFNxe2x80x9d) and individual Type I IFNs (Novick et al, 1995). A recombinant IFNAR2 subunit Ig fusion protein was shown to inhibit the binding of a variety of Type I IFN species (IFNxcex1A, IFNxcex1B, IFNxcex1D, IFNxcex2, IFNxcex1 Con1 and IFNxcfx89) to Daudi cells and xcex1/xcex2S subunit double transfected COS cells.
Type I IFN signaling pathways have recently been identified (Platanias et al, 1996; Yan et al, 1996; Qureshi et al, 1996; Duncan et al, 1996; Sharf et al, 1995; Yang et al, 1996). Initial events leading to signaling are thought to occur by the binding of IFNxcex1/xcex2/xcfx89 to the IFNAR2 subunit, followed by the IFNAR1 subunit associating to form an IFNAR1/2 complex (Platanias et al, 1994). The binding of IFNxcex1/xcex2/xcfx89 to the IFNAR1/2 complex results in the activation of two Janus kinases (Jak1 and Tyk2) which are believed to phosphorylate specific tyrosines on the IFNAR1 and IFNAR2 subunits. Once these subunits are phosphorylated, STAT molecules (STAT 1, 2 and 3) are phosphorylated, which results in dimerization of STAT transcription complexes followed by nuclear localization of the transcription complex and the activation of specific IFN inducible genes.
The pharmacokinetics and pharmacodynamics of Type I IFNs have been assessed in humans (Alan et al, 1997; Fierlbeck et al, 1996; Salmon et al, 1996). The clearance of IFNxcex2 is fairly rapid with the bioavailability of IFNxcex2 lower than expected for most cytokines. Although the pharmacodynamics of IFNxcex2 have been assessed in humans, no clear correlation has been established between the bioavailability of IFNxcex2 and clinical efficacy. In normal healthy human volunteers, administration of a single intravenous (iv) bolus dose (6 MIU) of recombinant CHO derived IFNxcex2 resulted in a rapid distribution phase of 5 minutes and a terminal half-life of xcx9c5 hours (Alam et al, 1997). Following subcutaneous (sc) or intramuscular (im) administration of IFNxcex2, serum levels are flat with only xcx9c15% of the dose systemically available. The pharmacodynamics of IFNxcex2 following iv, im or sc administration (as measured by changes in 2xe2x80x25xe2x80x2-oligoadenylate synthetase (2xe2x80x2,5xe2x80x2-AS) activity in PBMCs) were elevated within the first 24 hours and slowly decreased to baseline levels over the next 4 days. The magnitude and duration of the biologic effect was the same regardless of the route of administration.
The pharmacokinetics (PK) and pharmacodynamics (PD) of IFNxcex2 manufactured by two different companies (REBIF(copyright)-Serono and AVONEX(copyright)-Biogen) has been examined following the im injection of a single dose of 6 MIU of recombinant IFNxcex2 (Salmon, 1996). Serum concentration of IFNxcex2 and the IFNxcex2 surrogate marker, neopterin, were monitored over time. Both IFNxcex2 preparations exhibited similar PK profiles with peak serum levels of IFNxcex2 achieved by xcx9c12-15 hours, although REBIF(copyright) gave lower maximum levels. The IFNxcex2 levels remained elevated for both REBIF(copyright) and AVONEX(copyright) for at least the first 36 hours post im injection and then dropped to slightly above baseline by 48 hours. Levels of neopterin exhibited a very similar profile between REBIF(copyright) and AVONEX(copyright) with maximal neopterin levels achieved at xcx9c44-50 hours post-injection, remaining elevated until 72 hours post-injection and then dropping to baseline gradually by 144 hours.
A multiple dose pharmacodynamic study of IFNxcex2 has been conducted in human melanoma patients (Fierlbeck et al, 1996) with IFNxcex2 being administrated by sc route, three times per week at 3 MIU/dose over a six-month period. The pharmacodynamic markers, 2xe2x80x2, 5xe2x80x2-AS synthetase, xcex22-microglobulin, neopterin, and NK cell activation peaked by the second injection (day 4) and dropped off by 28 days, remaining only slightly elevated out to six months.
In summary, the clearance of Type I interferons in humans is rapid. A long-acting interferon preparation would likely result in an improvement in clinical benefit.
It has now been found that a Type I interferon complex, composed of soluble IFNAR complexed with Type I interferons (IFN), exhibits improved stability, enhanced potency, and elongated pharmacokinetics in vivo compared with free IFN for anti-viral, anti-cancer and immune modulating activity.
The present invention thus provides a Type I interferon (IFN) complex, composed of the polypeptide sequence of a human interferon xcex1/xcex2 receptor (IFNAR) subunit extracellular domain and Type I interferons, which exhibits improved stability, enhanced potency, and/or prolonged pharmacokinetics in vivo compared to free IFN for anti-viral, anti-cancer and immune modulating activity. Preferably, the complex is of the IFNAR2 subunit extracellular domain with any Type I interferon or the IFNAR1 subunit with IFNxcex1.
More specifically, the complex is a fusion protein, or a covalent complex, or a non-covalent complex containing the polypeptide sequence of the entire extracellular domain of IFNAR, preferably IFNAR2, or any interferon-binding subfraction thereof, complexed to IFNxcex1 or IFNxcex2 or IFNxcfx89, or any biologically active subfraction thereof.
IFNAR is intended to comprehend any of the known extracellular IFNAR receptors as defined above, as well as any active fragments thereof. IFNAR can be optionally fused to another protein, for example, an immunoglobulin such as IgG. IFN, IFNxcex1, IFNxcex2, and IFNxcfx89 are intended as one of the more than 20 Type I interferons identified to date, or any other Type I interferon identified in the future.
In one embodiment of the present invention, the complex is composed of IFNxcex1 or IFNxcex2, covalently linked to IFNAR2 via chemical linkage.
A further embodiment comprises a complex composed of IFNxcex1 or IFNxcex2, non-covalently complexed to IFNAR2. This further embodiment also includes a composition containing a Type I IFN and IFNAR2 in any ratio. A formulation of Type I IFN with an excess of IFNAR2 as defined above is also included in the definition of xe2x80x9ccomplexxe2x80x9d of the present application. The two components may also be administered separately so as to form the complex in vivo. Thus, in a further embodiment, the complex is a mixture of IFNAR2 and IFN, obtained by simultaneous or subsequent co-administration of IFNxcex1 or IFNxcex2 and soluble IFNAR2. Furthermore, the IFNAR can be administered without any concomitant administration of IFN, so that the complex may be formed in vivo with endogenous circulating IFN, thereby potentiating the effects of the endogenous IFN.
As a particular embodiment, the complex is composed of IFNxcex1 or IFNxcex2 or IFNxcfx89 fused to IFNAR2 as a recombinant fusion protein, where the IFN and the IFNAR2 moieties are optionally fused via a flexible peptide linker molecule. This peptide linker may or may not be cleavable in vivo.
The invention further relates to DNA encoding such fusion proteins, vectors containing such DNA, host cells transformed with such vectors in such a manner as to express the fusion proteins and methods of production of such fusion proteins by culturing such host cells and isolating the fusion proteins expressed thereby.
A further aspect of the present invention are the methods of use of the complexes of the present invention for prolonging the in vivo effect of IFN, which is useful in the treatment of any disease or condition which is treatable by IFN.
Another aspect of the present invention relates to the use of IFNAR as a stabilizer in formulations of IFN. Free IFNxcex2 has a tendency to oligomerize. This is prevented once it is complexed to IFNAR, particularly IFNAR2. Present day formulations of recombinant IFNxcex2 must have an acidic pH, which may cause some localized irritation when administered. Non-acidic compositions can be formulated if IFNAR is used as a stabilizer.